The goal of this Project is to develop assays and identify compounds that will be useful as research tools for studies of Mycobacterium tuberculosis (Mtb), and as potential drugs against tuberculosis (TB). It is anticipated that this approach will provide assay and chemical inputs into the pipeline for the development of new lead molecules for the treatment of TB infection, and for Mtb research. Compounds identified through the assay development and screening efforts of this Project, will be useful as research tools, providing important insights about the biological functions and structures of targeted Mtb membrane proteins, and will may be useful for therapeutic developments. This Project will focus on an Initial Target List of essential Mtb membrane proteins, selected for their functional importance, developed for this Program Project. The specific aims are (1) to develop biochemical assays for Mtb membrane proteins and perform high throughput screening of small molecule libraries, (2) to perform NMR screening of small molecule libraries with the Mtb proteins in micelles, and (3) to develop and perform NMR screening of small molecule libraries for the Mtb proteins in bilayers and bicelles. Biochemical assays and NMR-based methods for screening will be developed. The biochemical assays will facilitate further functional characterization, and enable the screening of compounds that interfere with activity. NMR will be used to screen for small molecules that bind to Mtb proteins even before a protein function is fully annotated. The identification of weak binders by this method is very useful for both the function and structure determination processes in Projects 1 and 3, and has the potential of identifying drug- like lead compounds for therapeutic development. NMR screening is well developed for globular proteins in aqueous solutions, but it has not been applied to membrane proteins in micelles or membrane proteins in lipid bilayers, and developing NMR screening for membrane proteins in lipid environments is a major goal of this Project.